Synthesis and structure activity relationships of glycine amide derivatives as novel Vascular Adhesion Protein-1 inhibitors

Article abstract of DOI:10.1016/j.bmc.2016.10.025

Vascular Adhesion Protein-1 (VAP-1) is a promising therapeutic target for the treatment of several inflammatory-related diseases including diabetic microvascular complication. We identified glycine amide derivative 3 as a novel structure with moderate VAP-1 inhibitory activity. Structure-activity relationship studies of glycine amide derivatives revealed that the tertiary amide moiety is important for stability in rat blood and that the position of substituents on the left phenyl ring plays an important role in VAP-1 inhibitory activity. We also found that low TPSA values and weak basicity are both important for high PAMPA values for glycine amide derivatives. These findings led to the identification of a series of orally active compounds with enhanced VAP-1 inhibitory activity. Of these compounds, 4g exhibited the most potent ex vivo efficacy, with plasma VAP-1 inhibitory activity of 60% after oral administration at 1 mg/kg.

Full text of DOI:10.1016/j.bmc.2016.10.025

Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and structure activity relationships of glycine amide
derivatives as novel Vascular Adhesion Protein-1 inhibitors
y
⇑
Susumu Yamaki , Daisuke Suzuki, Jiro Fujiyasu, Masahiro Neya , Akira Nagashima, Mitsuhiro Kondo,
Takafumi Akabane, Keitaro Kadono, Ayako Moritomo, Kosei Yoshihara à
Drug Discovery Research, Astellas Pharma Inc., 21 Miyukigaoka, Tsukuba, Ibaraki 305-8585, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Vascular Adhesion Protein-1 (VAP-1) is a promising therapeutic target for the treatment of several
inflammatory-related diseases including diabetic microvascular complication. We identified glycine
amide derivative 3 as a novel structure with moderate VAP-1 inhibitory activity. Structure-activity rela-
tionship studies of glycine amide derivatives revealed that the tertiary amide moiety is important for sta-
bility in rat blood and that the position of substituents on the left phenyl ring plays an important role in
VAP-1 inhibitory activity. We also found that low TPSA values and weak basicity are both important for
high PAMPA values for glycine amide derivatives. These findings led to the identification of a series of
orally active compounds with enhanced VAP-1 inhibitory activity. Of these compounds, 4g exhibited
the most potent ex vivo efficacy, with plasma VAP-1 inhibitory activity of 60% after oral administration
at 1 mg/kg.
Received 20 September 2016
Revised 18 October 2016
Accepted 19 October 2016
Available online xxxx
Keywords:
Vascular Adhesion Protein-1
Diabetic microvascular complication
Glycine amide
PAMPA
Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction
Increased plasma and/or membrane associated VAP-1 activities
have been found in patients with diabetic mellitus, and even more
Vascular Adhesion Protein-1 (VAP-1) is a member of the family
of copper-containing amine oxidases/semicarbazide-sensitive
amine oxidase (AOC/SSAO), found in humans as a membrane-
bound form and a soluble form. The membrane-bound form of
VAP-1 is mainly expressed in endothelial cells, smooth muscle
cells, and adipocytes, whereas the soluble VAP-1 is released into
plasma mainly from vascular endothelial cells.1
VAP-1 is reported to have two functions. As an adhesion mole-
cule, VAP-1 is involved in leukocyte rolling, adhesion and transmi-
gration, which are central steps in leukocyte extravasation to sites
of inflammation.2 Another function of VAP-1 is to act as an amine
oxidase. It possesses topaquinone (TPQ) in the active site as a
cofactor, and catalyzes the conversion of primary amines (e.g.,
methylamine and aminoacetone) into the corresponding aldehydes
(e.g., formaldehyde and methylglyoxal), while releasing ammonia
and hydrogen peroxide (Eq. (1)).3
so in patients with diabetic microvascular complication such as
diabetic retinopathy and diabetic nephropathy.4 Elevated expres-
sion of VAP-1 results in increased production of enzymatic prod-
ucts of VAP-1 like aldehyde and hydrogen peroxide. These
products have been reported to participate in the development of
diabetic microvascular complication, for example, by inducing
oxidative stress.5 Further, plasma and/or membrane bound VAP-1
is also increased, and is suggested to be involved in diseases such
as rheumatoid arthritis,6 atherosclerosis,7 chronic heart failure8
and Alzheimer’s disease.9 All of which are associated with inflam-
mation. These facts suggest that VAP-1 is a promising therapeutic
target for the treatment of several inflammatory-related diseases,
including diabetic microvascular complication.
Several approaches to inhibit VAP-1 have been reported, includ-
ing small interfering RNAs, function blocking antibodies, and small
molecule inhibitors.1,10 Among these, Bioite Therapeutics is con-
ducting clinical trials with their anti-VAP-1 antibody (BTT-1023)
for the treatment of autoimmune inflammatory and fibrotic dis-
RCH2NH2 þ O2 þ H2O ! RCHO þ H2O2 þ NH3
ð1Þ
eases.11 As for small molecule inhibitors, PXS-4728A (1) (IC50
=
5 nM12) has recently advanced to clinical trials for treatment of
non-alcoholic steatohepatitis (NASH) (Fig. 1).12
⇑
Corresponding author. Tel.: +81 29 863 6550; fax: +81 29 852 5387.
E-mail address: susumu.yamaki@astellas.com (S. Yamaki).
y
We have previously reported a novel VAP-1 inhibitor, com-
pound 2, which showed potent VAP-1 inhibitory activity with an
Present address: KNC Laboratories Co., Ltd, 1-1-1 Murotani, Nishiku, Kobe, Hyogo
651-2241, Japan.
à
IC50 value of 0.019 l
M (Fig. 1).13c Oral administration of this
Present address: Haupt Pharma Toride Co., Ltd, 5662 Omonmai, Toride, Ibaraki
302-0001, Japan.
http://dx.doi.org/10.1016/j.bmc.2016.10.025
0968-0896/Ó 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Yamaki, S.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.10.025

2
S. Yamaki et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
O
F
S
H
N
a, b
H
N
H
N
NH2
HN
NH2
N
NH2
O
NH2
O
O
N
·HCl
3
5
PXS-4728A (1)
2
Scheme 1. Reagents and conditions: (a) N-(tert-butoxycarbonyl)glycine,
WSCDꢀHCl, HOBt, DMF; (b) 4 M HCl (EtOAc solution).
Figure 1. Structures of PXS-4728A (1) and 2.
Scheme 4 shows the synthesis of compounds 17–19. Suzuki
coupling between aryl bromide 14 with 15 followed by deprotec-
tion of Boc group gave benzylamine 16. Benzylamine 16 was then
converted into 17–19 by condensation with the corresponding
amino acids and subsequent removal of Boc group.
The synthesis of 27–30 is shown in Scheme 5. Condensation of
benzylamine 6, 20 or isoindoline 22 with appropriate amino acids
afforded 23, 24 and 26. Substitution of benzylbromide 21 with tert-
butyl [2-(methylamino)ethyl]carbamate gave 25. Suzuki coupling
of the resulting 23–26 with 15 followed by deprotection of Boc
group gave 27–30.
Benzamide derivative 33 was synthesized as shown in
Scheme 6. Suzuki coupling between aryl bromide 31 and 15
followed by hydrolysis using NaOH gave carboxylic acid 32. Con-
densation of 32 with tert-butyl [2-(methylamino)ethyl]carbamate
followed by removal of Boc group gave compound 33.
compound at 10 mg/kg showed an inhibitory effect on ocular
permeability in streptozotocin (STZ)-induced diabetic rats. These
results suggested that VAP-1 would be a promising therapeutic
target for the treatment of diabetic macular edema. However,
this compound was not suitable for further clinical development
due to its insufficient pharmacokinetic properties in rats
(F = 0.7%). These facts prompted us to identify novel VAP-1
inhibitors.
To find a novel class of orally active VAP-1 inhibitors, we
screened our in-house compound library for VAP-1 enzymatic inhi-
bitory activity and identified compound 3 as a moderate VAP-1
inhibitor with an IC50 value of 0.18 lM (Table 1). Although this
compound possesses a novel scaffold with moderate VAP-1 inhibi-
tory activity, 3 showed poor ex vivo efficacy (17% inhibition in rat
plasma VAP-1 activity after oral administration at 100 mg/kg). We
therefore conducted structural optimization of 3 in order to
improve its ex vivo efficacy. In this manuscript, we describe the
synthesis and structure-activity relationships of glycine amide
derivatives as novel VAP-1 inhibitors.
3. Results and discussion
The inhibitory activities of the synthesized compounds against
human and rat VAP-1 were measured by a radiochemical-enzyme
assay using 14C-benzylamine as an artificial substrate. Plasma VAP-
1 activities in normal rats after oral administration of test com-
pounds were evaluated by using the same radiochemical-enzyme
assay.
We identified glycine amide derivative 3 as a novel structure
from our in-house compound library. This compound showed
moderate VAP-1 inhibitory activity with an IC50 value of
2. Chemistry
The synthesis of glycine amide derivatives is outlined in
Schemes 1–6. Condensation of benzylamine 5 with N-(tert-butoxy-
carbonyl)glycine followed by deprotection of tert-butoxycarbonyl
(Boc) group gave 3 (Scheme 1). Scheme 2 shows the synthesis of
4a–4h. Condensation of benzylamine 6 with N-Boc-glycine gave
7. Suzuki coupling of 7 with various boronic acids or esters fol-
lowed by removal of Boc group afforded 4a–4h.
0.18 lM. However, it showed poor ex vivo efficacy (17% inhibition
in rat plasma VAP-1 activity after oral administration at 100 mg/
kg) in spite of the fact that there were no particular problems with
solubility, membrane permeability, or metabolic stability in liver
microsomes (data not shown). Since an amide bond would be
sometimes vulnerable to hydrolysis, we suspected the stability of
this compound in vivo and examined its stability in rat blood. As
shown in Figure 2, it was found that concentration of 3 was
decreased to 33% after incubation with rat blood for 1 h. Therefore,
we considered that instability of 3 in rat blood is one of the reasons
for low ex vivo activity. Because we needed to confirm the activity
of compounds in rodent, such as rat, before advancing to clinical
trial, we considered it important to improve stability in rat blood
to obtain a compound showing potent ex vivo efficacy. Testa
Compounds 10, 11a–11d, 12a–12b, and 13 were prepared by
the synthetic scheme depicted in Scheme 3. Suzuki coupling
between 7 and corresponding boronic acids or esters gave biphenyl
analogs 8a–8c. Hydrolysis and removal of Boc group of 8a by treat-
ment with 6 M HCl at 100 °C yielded 10. Hydrolysis of 8a by NaOH
gave carboxylic acid 9, which was then converted into amide
derivatives 11a–11d by condensation with a variety of amines
and subsequent deprotection of Boc group. Treatment of aniline
8b with carbamoyl chlorides followed by removal of Boc group
yielded 12a–12b respectively. Reductive amination of aldehyde
8c with diethylamine followed by removal of Boc group gave ben-
zylamine analog 13.
Table 1
In vitro and pharmacodynamic profile of 3
Compound
Structure
Human VAP-1 IC50
(
l
M)a
Rat ex vivob
Inhibition ratio (at 1 h) (%)
Dose (mg/kg, po)
100
H
N
3c
0.18
17
NH2
O
a
b
c
IC50 value is shown as the mean of independent experiments (n = 2).
Inhibitory effect on plasma VAP-1 activity in rats (n = 5) at 1 h after oral administration of compound 3.
Hydrochloride salt.
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S. Yamaki et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
3
2
b, c
a
N
N
3
4
NH2
NH
Br
NHBoc
Br
O
O
R
6
7
4a: R = H
4b: R = 4-Cl
4c: R = 3-Cl
4d: R = 2-Cl
4e: R = 4-NHAc
4f: R = 4-N(Me)2
4g: R = 4-morpholyl
4h: R = 4-(4-methyl)-piperazyl
Scheme 2. Reagents and conditions: (a) N-(tert-butoxycarbonyl)glycine, WSCDꢀHCl, HOBt, CH2Cl2; (b) Ar-B(OH)2 or Ar-B(Pin), Pd(PPh3)4, Na2CO3, solvents, 80 °C; (c) 4 M HCl
(EtOAc solution), EtOAc.
et al. reported that a tertiary amide would be more stable than the
secondary amide against hydrolysis in the case of amides with
bulky substituents on nitrogen.14 Thus, we investigated the effect
of conversion from secondary amide into tertiary amide on the sta-
bility in rat blood. As expected, tertiary amide derivative 4a was
found to be more stable in rat blood compared to 3, with no appar-
ent decrease in 4a was observed even after incubation with rat
blood for 6 h (Fig. 2). However, in vitro potency of 4a to both
human and rat VAP-1 enzyme were decreased more than 4-fold
as shown in Table 2. In spite of its decreased in vitro potency, 4a
exhibited more potent ex vivo efficacy than 3 (3: 17% inhibition
at 100 mg/kg, 4a: 69% inhibition at 60 mg/kg). We speculated that
4a has a higher plasma concentration than 3 because of its
improved stability in rat blood. These results suggested that con-
version to the tertiary amide from the secondary amide is impor-
tant for stability in rat blood and results in potentiation of
ex vivo efficacy. We expected that an improvement in the VAP-1
inhibitory activity of 4a while maintaining stability in rat blood
would lead to a compound with more potent ex vivo activity. Thus,
(human IC50 = 0.80
l
M, rat IC50 = 0.12
lM) compared to that of 3
N
NH2
O
HO2C
b
c
10
a
N
NHBoc
d, e
N
N
O
NH2
NHBoc
MeO C
2
O
O
R
8a
HO2C
11a: R = CONH2
11b: R = CONHEt
11c: R = CONEt2
9
f, e
O
a
a
N
N
O
NHBoc
O
NH2
N
N
11d: R =
Br
NHBoc
O
H2N
R
N
H
O
O
8b
7
N
N
12a: R =
12b: R =
O
g, e
N
N
NHBoc
NH2
O
N
O
OHC
8c
13
Scheme 3. Reagents and conditions: (a) Ar-B(OH)2 or Ar-B(Pin), Pd(PPh3)4, Na2CO3, DME/EtOH/H2O, 80 °C; (b) 6 M HCl aq, 100 °C; (c) 1 M NaOH aq, MeOH; (d) amine,
WSCDꢀHCl or WSCD, HOBt, with or without Et3N, DMF; (e) 4 M HCl (EtOAc solution), solvents; (f) carbamoyl chloride, base, CH2Cl2; (g) diethylamine, NaBH(OAc)3, AcOH, DCE.
N
R1
NH
c or c, b
a, b
N
R2
n
NBoc
N
O
Br
N
N
O
O
14
16
17: R1 = Me, R2 = H, n = 1
18: R1 = R2 = Me, n = 1
19: R1 = R2 = H, n =2
O
BPin
15
Scheme 4. Reagents and conditions: (a) 15, Pd(PPh3)4, Na2CO3, DME/H2O, 90 °C; (b) 4 M HCl (EtOAc solution), EtOH; (c) amino acids, WSCDꢀHCl, HOBt, solvents.
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S. Yamaki et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
R
c, d
a or b
R
R
Br
Br
N
O
6: R = NHMe
20: R = NHCH(Me)2
21: R = Br
N
N
23: R =
27: R =
NHBoc
NH2
O
O
O
O
N
N
N
N
24: R =
25: R =
28: R =
29: R =
NHBoc
NHBoc
NH2
NH2
NH2
N
a
NHBoc
c, d
O
NH
N
O
Br
Br
N
O
22
26
30
Scheme 5. Reagents and conditions: (a) amino acids, WSCDꢀHCl, HOBt, CH2Cl2; (b) tert-butyl [2-(methylamino)ethyl]carbamate, K2CO3, DMF; (c) 15, Pd(PPh3)4, Na2CO3, DME/
H2O, 80–90 °C; (d) 4 M HCl (EtOAc solution), solvents.
a, b
c, d
N
NH2
CO2H
Br
CO2Me
O
N
N
O
O
31
32
33
Scheme 6. Reagents and conditions: (a) 15, Pd(PPh3)4, Na2CO3, DME/H2O, 80 °C (b) 1 M NaOH aq, EtOH, 60 °C; (c) tert-butyl [2-(methylamino)ethyl]carbamate, WSCDꢀHCl,
HOBt, DMF; (d) 4 M HCl (EtOAc solution), MeOH.
we planned further modification of 4a in order to increase in vitro
potency.
To design more potent VAP-1 inhibitors, we initially performed
docking analysis of 4a with the human VAP-1 model using Inoue’s
method, as shown in Figure 3.13 As a starting structure, the primary
amine group of 4a formed a Schiff base intermediate with TPQ. In
addition to this covalent interaction of the primary amine group,
this model revealed the following three interactions. First, the left
phenyl ring of 4a formed a CH-
the oxygen of the carbonyl group formed a CH-O interaction with
Leu468. Third, the hydrogen at the -position of the carbonyl
p interaction with Leu447. Second,
a
group formed a CH-O interaction with the Leu468 backbone car-
bonyl oxygen. Since the glycine amide moiety (right part) and left
phenyl ring interact with VAP-1, we considered that the structure
of the right part and the spatial relationship between the left phe-
nyl ring and the right part are important for VAP-1 inhibitory activ-
Figure 2. Residual ratios (% to initial concentration) of compound 3 and 4a after
incubation with rat blood (n = 3). Hydrochloride salt.
a
Table 2
In vitro and pharmacodynamic profile of 3 and 4a
R
N
NH2
O
Compound
R
VAP-1 IC50
(l
M)a
Rat ex vivob
Human
Rat
Dose (mg/kg, po)
Inhibition ratio (at 1 h) (%)
3c
4a
H
Me
0.18
0.80
0.023
0.12
100
60
17
69
a
b
c
IC50 values are shown as the mean of independent experiments (n = 2–3).
Inhibitory effect on plasma VAP-1 activity in rats (n = 3–5) at 1 h after oral administration of compound 3 or 4a.
Hydrochloride salt.
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S. Yamaki et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
5
Table 3
(a)
(b)
SARs of chloro group substituted derivatives
Central phenyl ring
2
N
NH2
3
4
R
O
Compound
R
Human VAP-1 IC50 (l
M)a
4a
Right part
4a
H
0.80
0.24
1.3
Left phenyl ring
4bb
4cc
4db
4-Cl
3-Cl
2-Cl
4.1
a
b
c
IC50 value is shown as the mean of independent experiments (n = 2–3).
Hydrochloride salt.
Ethanedioate salt.
decreased inhibitory activity compared to 4a. These SARs sug-
gested that introduction of substituents at the 4-position of the left
phenyl ring is optimal for the improvement of VAP-1 inhibitory
activities. Based on this result, we next investigated the effect of
several substituent groups at the 4-position on the left phenyl ring.
Polar substituents were considered for introduction as there are
hydrophilic residues like Asp446 and His450 around the solvent
side.
Table 4 shows VAP-1 inhibitory activities of compounds having
carboxylic acid, amide and urea substituent groups at the 4-posi-
tion on the left phenyl ring. Introduction of carboxylic acid resulted
in a loss of VAP-1 inhibitory activity (10), suggesting that anionic
groups are not tolerated at this position. While carbamoyl analog
(c)
11a showed 2-fold decreased potency (IC50 = 1.6 lM) compared
to 4a, all the other compounds in Table 4 (11b–11d, 4e, and
12a–12b), which have an amide or urea moiety, exhibited
increased inhibitory activity (IC50 = 0.083–0.61
12b exhibited the most potent VAP-1 inhibitory activity with an
IC50 value of 0.083 M among the compounds we examined. These
lM). In particular,
l
results were consistent with the results of docking analysis, and
suggested that steric hindrance at this position is well tolerated.
Parallel artificial membrane permeability assay (PAMPA) is a
commonly used model to predict the permeability of a compound
by measuring its flux across an artificial membrane.15 11b–11d, 4e,
and 12a–12b showed low permeability in PAMPA (Pe -
6 3.8 ꢁ 10ꢂ6 cm/s). Since these results predicted the possibility of
poor membrane permeability of these compounds leading to poor
absorption from the small intestine, we considered that it is impor-
tant to improve permeability in PAMPA. PAMPA value often corre-
lates with topological polar surface area (TPSA),15b and compounds
in Table 4 tend to show this correlation. TPSA values were
calculated using Molecular Operating Environment (MOE).16b The
values of 11b–11d, 4e, and 12a–12b which showed poor PAMPA
values were higher than 60 Å2, whereas 4a and 4b showed good
PAMPA values (Pe > 30 ꢁ 10ꢂ6 cm/s) with lower TPSA value
(46.3 Å2) than 60 Å2. Thus, we designed a compound with a TPSA
value lower than 60 Å2 to improve permeability in PAMPA.
Figure 3. (a) Structure of 4a. (b) Molecular modeling results for 4a with human
VAP-1. (c) Two-dimensional diagram prepared by the ligand interactions applica-
tion in MOE.16b Arrows indicate interaction.
ity. In addition to these interactions, this molecular modeling pre-
dicted that there is limited steric tolerance around the central phe-
nyl ring and right part since these sites are contiguous with amino
acid residues such as Leu468, Leu469, Met211, and Thr212. In con-
trast to the central phenyl ring and right part, considerable space
around the 4-position of the left phenyl ring is suggested since this
side faces toward the solvent side. The results of docking analysis
of 4a suggested that it is most reasonable to introduce substituents
around the 4-position of the left phenyl ring in order to improve
VAP-1 inhibitory activity of 4a.
The results of benzylamine and aniline analogs which have
TPSA values lower than 60 Å2 are summarized in Table 5. Benzy-
lamine derivative 13 showed improved VAP-1 inhibitory activity
(IC50 = 0.48 lM) compared to 4a, indicating that introduction of a
basic substituent group at this position is also well tolerated.
Based on the results from the docking analysis mentioned
above, we introduced chloro group into the 4-position of the left
phenyl ring of 4a (Table 3). Predictably, introduction of chloro
group at this position was well tolerated, as 4-chloro derivative
4b showed 3-fold increased inhibitory activity compared to 4a
Although dimethylamino analog 4f showed decreased activity
(IC50 = 3.0
lM), morpholino analog 4g and N-methyl-piperazino
analog 4h showed improved VAP-1 inhibitory activity (IC50 = 0.34
and 0.33 lM, respectively) compared to 4a. As we expected, 4f
and 4g, which had TPSA values lower than 60 Å2 (49.6 and
58.8 Å2, respectively) showed improved PAMPA values (Pe = 28.1
and 22.1 ꢁ 10ꢂ6 cm/s, respectively). On the other hand, 13 and
with an IC50 value of 0.24
lM. In contrast, 3-chloro analog 4c
(IC50 = 1.3 M) and 2-chloro analog 4d (IC50 = 4.1
l
lM) showed
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6
S. Yamaki et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
Table 4
SARs of 4-substituted derivatives
N
NH2
O
R
Compound
R
Human VAP-1 IC50
(
l
M)a
PAMPAb, Pe (10ꢂ6 cm/s) at pH = 6.5
TPSA (Å2)c
4a
H
Cl
0.80
0.24
14
1.6
0.61
0.56
>30
>30
NTg
NTg
<0.2
3.8
46.3
46.3
83.6
89.4
75.4
66.6
4bd
10d
HO2C–
H2NOC–
EtHNOC–
Et2NOC–
11ad
11bd
11ce
O
11df
4ed
0.42
0.46
0.28
<0.1
<0.2
<0.2
75.9
75.4
78.7
N
O
AcHN–
O
12ae
N
N
H
O
12be
0.083
<0.2
87.9
N
N
H
O
a
b
c
d
e
f
IC50 value is shown as the mean of independent experiments (n = 2–3).
PAMPA EvolutionTM (pION Inc., Billerica, MA), donor buffer pH = 6.5.
TPSA values calculated by MOE2014.0901.
Hydrochloride salt.
Ethanedioate salt.
(2R,3R)-Tartrate salt.
g
NT = not tested.
Table 5
SARs of benzylamine and aniline derivatives
N
NH2
O
R
M)a
PAMPAb, Pe (10ꢂ6 cm/s) at pH = 6.5
TPSA (Å2)c
pKa
d
Compound
R
Human VAP-1 IC50
(l
4a
H
0.80
0.48
3.0
>30
1.0
28.1
22.1
46.3
49.6
49.6
58.8
—
13e
4ff
4gf
Et2NH2C–
Me2N–
Morpholine
9.7
5.0
5.1
0.34
N
N
4hg
0.33
1.0
52.8
7.8 3.1
a
b
c
d
e
f
IC50 value is shown as the mean of independent experiments (n = 2–3).
PAMPA EvolutionTM (pION Inc., Billerica, MA), donor buffer pH = 6.5.
TPSA values calculated by MOE2014.0901.
pKa values other than the primary amine group which were calculated using ACD pKa prediction software.17
Dihydrochloride salt.
Ethanedioate salt.
(2R,3R)-Tartrate salt.
g
4h showed poor PAMPA values (Pe = 1.0 ꢁ 10ꢂ6 cm/s) in spite of
having lower TPSA values (49.6 and 52.8 Å2, respectively) than
60 Å2. These poor PAMPA values of 13 and 4h may be due in part
to the strong basicity of benzylamine (pKa = 9.7, calculated using
ACD pKa prediction software17) in 13 and N-methyl-piperazine
(pKa = 7.8) in 4h. These results from Table 5 suggested that low
TPSA values (lower than 60 Å2) and weak basicity (lower than
5.1) are both important for high PAMPA values of glycine amide
derivatives. Among the compounds examined in Table 5, 4g exhib-
ited an improvement in in vitro potency with high PAMPA value.
We next investigated the SARs around the right part of 4g as
summarized in Table 6. Conversion of the primary amine group
into a secondary amine group (17) or tertiary amine group (18)
resulted in a loss of VAP-1 inhibitory activity. These results sug-
gested that the primary amine group is essential for VAP-1 inhibi-
tory activity. This is consistent with our assumption that the
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S. Yamaki et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
7
Table 6
group (33) showed less potent VAP-1 inhibitory activities com-
pared to 4g, suggesting that the position and existence of the car-
bonyl group are also important for VAP-1 inhibitory activity. As
predicted from the results of docking analysis, these results in
Table 6 indicated that conversion of the right part in 4g while
maintaining VAP-1 inhibitory activity is difficult.
The results of docking analysis of 4g with the human VAP-1
model are shown in Figure 4. The predicted binding mode of 4g
was similar to that of 4a. Introduction of morpholine at the 4-posi-
tion of the left phenyl ring might have contributed to increased
inhibitory activity due to increased Van der Waals interaction by
occupying the solvent site.
SARs of 4-(biphenyl-4-yl)morpholine derivatives
R
N
O
Compound R or structure
Human VAP-1
IC50 (l
M)a
N
The inhibitory activities of 4b and 4g against rat VAP-1 were
further evaluated using a radiochemical enzyme assay (Table 7).
4b and 4g showed more potent rat VAP-1 inhibitory activities with
4gb
0.34
NH2
O
N
an IC50 value of 0.053
lM and 0.036 lM, respectively, compared to
17c
18c
27c
N
H
>100
>100
>100
4a (IC50 = 0.12 M). On the basis of enhanced in vitro activity with
l
high PAMPA values (Pe > 20 ꢁ 10ꢂ6 cm/s), 4b and 4g were selected
for further evaluation (Table 7). Oral administration of 4b and 4g at
10 mg/kg inhibited rat plasma VAP-1 activity by 35% and 92%,
respectively. Furthermore, 4g exhibited a VAP-1 inhibitory effect
in rat plasma from lower dose, as oral administration of 4g at
1 mg/kg inhibited rat plasma VAP-1 activity by 60%. In addition
to the improved in vitro potency of 4g, the reason for the potent
ex vivo efficacy of this compound may be explained in part by
O
O
O
N
N
N
NH2
28c
>100
2.0
N
NH2
O
O
N
NH2
30d
N
N
O
NH2
19c
29c
33c
4.0
O
N
N
1.8
NH2
NH2
>100
O
a
b
c
IC50 value is shown as the mean of independent experiments (n = 2).
Ethanedioate salt.
(2R,3R)-Tartrate salt
Hydrochloride salt.
d
primary amine group would form a Shiff base intermediate with
TPQ to inhibit VAP-1 activity. 27 (gem-dimethyl derivative)
showed no activity, this may be responsible for the difficulty of
27 in forming a Shiff base intermediate with TPQ due to its steric
hindrance adjacent to the primary amine group. Introduction of
an isopropyl group on the nitrogen atom (28) also resulted in a loss
of VAP-1 inhibitory activity. This result suggested that steric hin-
drance around the nitrogen atom is very limited. Isoindoline ana-
log 30 showed 6-fold decreased inhibitory activity compared to
4g. The reason for this may be that 30 could not interact properly
with VAP-1 enzyme due to an altered spatial relationship between
the left phenyl ring and right part because of its cyclic structure.
Introduction of a longer alkyl chain (19) led to deceased VAP-1
inhibitory activity (IC50 = 4.0 lM), suggesting that length of the
alkyl chain between the carbonyl group and primary amine group
is also important for VAP-1 inhibitory activity. Removal of the car-
bonyl group (29) and conversion of the position of the carbonyl
Figure 4. (a) Molecular modeling results for 4a (pink) and 4g (yellow, ball and
stick) with human VAP-1. (b) Two-dimensional diagram prepared by the ligand
interactions application in MOE.16b Arrows indicate interaction.
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8
S. Yamaki et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
Table 7
Rat VAP-1 inhibitory activities and pharmacodynamic profiles of 4a, 4b, and 4g
N
NH2
O
R
Compound
R
VAP-1 IC50
Human
(
l
M)a
Rat
Rat ex vivob
Inhibition ratio (at 1 h) (%)
CLogPc
Dose (mg/kg, po)
4a
H
Cl
0.80
0.24
0.34
0.12
0.053
0.036
60
10
10
3
69
35
92
87
60
2.08
2.65
1.22
4bd
4ge
Morpholine
1
a
b
c
IC50 values are shown as the mean of independent experiments (n = 2–3).
Inhibitory effect on plasma VAP-1 activity in rats (n = 3–5) at 1 h after oral administration of test compounds.
LogP values calculated using ACD LogP prediction software.17
Hydrochloride salt.
d
e
Ethanedioate salt.
Table 8
Pharmacokinetic profiles of compound 4g in rats
Route
AUC0–24h (ngꢀh/mL)
126.7
69.6
Cmax (ng/mL)
CLtot (mL/min/kg)
133.9
t1/2 (h)
Vdss (L/kg)
BA (%)
54.9
iv (1 mg/kg)
po (1 mg/kg)
0.47
0.58
6.06
73.4
lower plasma protein binding ratio of 4g (4a: 71%, 4b: 92%, 4g:
62%) due to its lower lipophilicity (CLogP values; 4a: 2.08, 4b:
2.65, 4g: 1.22, calculated using ACD LogP prediction software17),
which may lead to a higher unbound plasma concentration of 4g
compared to 4a and 4b.
4g showed an acceptable pharmacokinetic profile as shown in
Table 8. Plasma Cmax was 73.4 ng/mL at an oral dose of 1 mg/kg,
which is 6 times higher than the IC50 value of 4g (0.036 lM, rat).
mier mass spectrometer. Elemental analyses were conducted using
a Yanaco MT-6 (C, H, N), Yanaco JM10 (C, H, N), Elementar Vario EL
III (C, H, X), Dionex ICS-3000 (S, halogene), and Dionex DX-500 (S,
halogene) and were within 0.4% of theoretical values. All reac-
tions were carried out using commercially available reagents and
solvents without further purification. The following abbreviations
are used: AcOH, acetic acid; MeCN, acetonitrile; DCE, 1,2-dichlor-
oethane; DME, 1,2-dimethoxyethane; DMF, N,N-dimethylfor-
mamide; DMSO, N,N-dimethylsulfoxide; EtOAc, ethyl acetate;
EtOH, ethanol; Et2O, diethyl ether; HOBt, 1-hydroxybenzotriazole;
MeOH, methanol; WSCD, 1-ethyl-3-(30-dimethylaminopropyl) car-
bodiimide; and Et3N, triethylamine.
4. Conclusion
In summary, we synthesized and evaluated a series of glycine
amide derivatives as novel VAP-1 inhibitors, and revealed that
the tertiary amide moiety is important for stability in rat blood.
Based on the docking analysis, we also found that the 4-position
of the left phenyl ring of glycine amide derivatives is an optimal
position for introducing substituent groups to increase VAP-1 inhi-
bitory activity. In addition, we found that, for glycine amide deriva-
tives, low TPSA values and weak basicity are both important for
good PAMPA values. These findings led to the identification of 4b
(chloro analog) and 4g (morpholino analog), which had enhanced
VAP-1 inhibitory activity while maintaining good PAMPA values.
Further, these compounds were subjected to rat ex vivo assay,
and 4g showed the most potent inhibitory effect on rat plasma
VAP-1 activity after oral administration. Further studies to identify
potent and orally available VAP-1 inhibitors for clinical develop-
ment are underway.
5.1.1. N-(Biphenyl-3-ylmethyl)glycinamide hydrochloride (3)
To a solution of 3-phenylbenzylamine (5; 190 mg, 1.04 mmol),
N-(tert-butoxycarbonyl)glycine (200 mg, 1.14 mmol) and HOBt
(154 mg, 1.14 mmol) in DMF was added WSCDꢀHCl (219 mg,
1.14 mmol) at room temperature. After being stirred overnight,
the mixture was concentrated in vacuo. The residue was diluted
with EtOAc and the mixture was washed with 0.5 M HCl aqueous
solution, 1 M NaHCO3 aqueous solution, and brine. The organic
layer was dried over MgSO4 and concentrated in vacuo to give
the solid. This solid was dissolved in 4 M HCl/EtOAc at room tem-
perature. After being stirred for 1 h, the mixture was concentrated
in vacuo. The residue was triturated with Et2O and filtered to give
the product (285 mg, 99%) as a solid. 1H NMR (DMSO-d6) d 3.63
(2H, s), 4.43 (2H, d, J = 5.8 Hz), 7.30 (1H, d, J = 7.8 Hz), 7.35–7.51
(4H, m), 7.54–7.62 (2H, m), 7.64–7.69 (2H, m), 8.18 (3H, br s),
9.02 (1H, t, J = 5.6 Hz); MS (ESI) m/z [M+H]+ 241; Anal. Calcd for
5. Experimental
5.1. Chemistry
C15H16N2OꢀHClꢀ0.2H2O: C, 64.26; H, 6.26; N, 9.99; Cl, 12.64. Found:
C, 64.47; H, 6.21; N, 10.14; Cl, 12.43.
5.1.2. tert-Butyl {2-[(3-bromobenzyl)(methyl)amino]-2-
oxoethyl}carbamate (7)
To a solution of 1-(3-bromophenyl)-N-methylmethanamine (6;
45.6 g, 228 mmol) in CH2Cl2 (700 mL) were added N-(tert-butoxy-
1H NMR spectra were recorded on a Varian VNS-400, JEOL JNM-
LA400 or JEOL JNM-AL400 spectrometer. Chemical shifts were
expressed in d values (ppm) using tetramethylsilane as the internal
standard (s = singlet, d = doublet, t = triplet, m = multiplet,
dd = double doublet, and br = broad peak). Mass spectra (MS) were
recorded on a JEOL LX-2000, Waters ZQ-2000 or Waters LCT Pre-
carbonyl)glycine
(43.9 g,
251 mmol),
WSCDꢀHCl
(52.4 g,
274 mmol), and HOBt (37.1 g, 275 mmol). After being stirred at
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S. Yamaki et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
9
room temperature for 1 week, the mixture was diluted with water
and the mixture was extracted with CHCl3. The organic layer was
washed with saturated NaHCO3 aqueous solution and brine, dried
over Na2SO4, and concentrated in vacuo. The residue was purified
by column chromatography on silica gel (hexane/EtOAc = 7:1 to
1:1) to give the product (49.1 g, 60%) as an oil. 1H NMR (CDCl3):
this compound exists as a pair of rotamers at room temperature.
d 1.44 (minor rotamer, 9H, s), 1.46 (major rotamer, 9H, s), 2.90
(major rotamer, 3H, s), 2.98 (minor rotamer, 3H, s), 3.96–4.06
(2H, m), 4.44 (minor rotamer, 2H, s), 4.57 (major rotamer, 2H, s),
5.54 (1H, br s), 7.06–7.25 (2H, m), 7.28–7.49 (2H, m); MS (ESI)
m/z [M+H]+ 357, 359.
(192 mg, 1.23 mmol), Na2CO3 (174 mg, 1.64 mmol), and Pd
(PPh3)4 (28 mg, 0.025 mmol). The mixture was stirred at 80 °C
overnight. After being cooled to room temperature, the mixture
was diluted with water and extracted with EtOAc. The organic
layer was dried over MgSO4 and concentrated in vacuo. The residue
was purified by column chromatography on silica gel (hexane/
EtOAc = 1:0 to 1:2) to afford tert-butyl (2-{[(30-chlorobiphenyl-3-
yl)methyl](methyl)amino}-2-oxoethyl)carbamate. To a solution of
tert-butyl (2-{[(30-chlorobiphenyl-3-yl)methyl](methyl)amino}-2-
oxoethyl)carbamate in EtOAc (5.0 mL) was added 4 M HCl/EtOAc
(10.0 mL, 40.0 mmol). After being stirred at room temperature for
2 h, the mixture was concentrated in vacuo. The residue was
diluted with saturated NaHCO3 aqueous solution and the mixture
was extracted with CHCl3. The organic layer was dried over Na2SO4
and concentrated in vacuo. To a solution of the residue in EtOH was
added oxalic acid (74 mg, 0.82 mmol). The mixture was concen-
trated in vacuo to give the product (186 mg, 60%) as a colorless
solid. 1H NMR (DMSO-d6): this compound exists as a pair of rota-
mers at room temperature. d 2.90 (minor rotamer, 3H, s), 2.96
(major rotamer, 3H, s), 3.93 (minor rotamer, 2H, s), 3.96 (major
rotamer, 2H, s), 4.61 (minor rotamer, 2H, s), 4.64 (major rotamer,
2H, s), 7.27–7.34 (1H, m), 7.44–7.67 (6H, m), 7.71 (major rotamer,
1H, dd J = 1.8, 1.8 Hz), 7.74 (minor rotamer, 1H, dd J = 1.8, 1.8 Hz);
MS (FAB) m/z [M+H]+ 289; Anal. Calcd for C16H17ClN2OꢀC2H2O4: C,
57.07; H, 5.06; N, 7.40; Cl, 9.36. Found: C, 57.03; H, 5.04; N, 7.37;
Cl, 9.40.
5.1.3. N-(Biphenyl-3-ylmethyl)-N-methylglycinamide (4a)
To a mixture of 7 (215 mg, 0.60 mmol) in dioxane (4.0 mL)/
water (1.0 mL) were added phenylboronic acid (110 mg,
0.90 mmol), Na2CO3 (200 mg, 1.89 mmol), and Pd(PPh3)4 (33 mg,
0.029 mmol) under an argon atmosphere. The mixture was stirred
at 80 °C for 16 h. After being cooled to room temperature, the mix-
ture was diluted with water and extracted with EtOAc. The organic
layer was dried over MgSO4 and concentrated in vacuo. The residue
was purified by column chromatography on silica gel (hexane/
EtOAc = 98:2 to 2:1) to afford tert-butyl {2-[(biphenyl-3-
ylmethyl)(methyl)amino]-2-oxoethyl}carbamate. To a solution of
tert-butyl {2-[(biphenyl-3-ylmethyl)(methyl)amino]-2-oxoethyl}-
carbamate in EtOAc (3.0 mL) was added 4 M HCl/EtOAc (2.00 mL,
8.00 mmol). After being stirred at room temperature for 2 h, the
resulting precipitate was filtered to give N-(Biphenyl-3-
ylmethyl)-N-methylglycinamide hydrochloride (146 mg, 83%) as
a colorless solid. 1H NMR (DMSO-d6): this compound exists as a
pair of rotamers at room temperature. d 2.91 (minor rotamer, 3H,
s), 2.97 (major rotamer, 3H, s), 3.91 (minor rotamer, 2H, s), 3.96
(major rotamer, 2H, s), 4.63 (minor rotamer, 2H, s), 4.64 (major
rotamer, 2H, s), 7.24–7.29 (1H, m), 7.36–7.42 (1H, m), 7.42–7.51
(3H, m), 7.52–7.70 (4H, m), 8.16 (3H, br s); MS (ESI) m/z [M+H]+
255; Anal. Calcd for C16H18N2OꢀHCl: C, 66.09; H, 6.59; N, 9.63; Cl,
12.19. Found: C, 66.10; H, 6.63; N, 9.60; Cl, 11.96.
To a solution of N-(Biphenyl-3-ylmethyl)-N-methylglycinamide
hydrochloride (240 mg, 0.83 mmol) in water (5.0 mL) was added 1
M NaHCO3 aqueous solution until pH = 8. The mixture was extracted
with CHCl3. The organic layer was washed with brine and concen-
trated in vacuo to give the product (200 mg, 95%) as a solid. 1H
NMR (DMSO-d6): this compound exists as a pair of rotamers at room
temperature. d 1.74 (2H, br s), 2.87 (minor rotamer, 3H, s), 2.89
(major rotamer, 3H, s), 3.40 (2H, s), 4.58 (minor rotamer, 2H, s),
4.59 (major rotamer, 2H, s), 7.17–7.26 (1H, m), 7.35–7.52 (5H, m),
7.52–7.59 (1H, m), 7.62–7.67 (2H, m); MS (FAB) m/z [M+H]+ 255.
5.1.6. N-[(20-Chlorobiphenyl-3-yl)methyl]-N-methylglycinamide
hydrochloride (4d)
Compound 4d was prepared from 7 and (2-chlorophenyl)boro-
nic acid in 73% yield as a colorless solid, using a similar approach to
that described for 4aꢀHCl. 1H NMR (DMSO-d6): this compound
exists as a pair of rotamers at room temperature. d 2.91 (minor
rotamer, 3H, s), 2.96 (major rotamer, 3H, s), 3.88 (minor rotamer,
2H, s), 3.94 (major rotamer, 2H, s), 4.63 (2H, s), 7.28–7.53 (7H,
m), 7.56–7.60 (1H, m), 8.23 (3H, br s); MS (FAB) m/z [M+H]+ 289;
Anal. Calcd for C16H17ClN2OꢀHCl: C, 59.09; H, 5.58; N, 8.61; Cl,
21.80. Found: C, 59.08; H, 5.62; N, 8.67; Cl, 21.65.
5.1.7. N-[(40-Acetamidobiphenyl-3-yl)methyl]-N-
methylglycinamide hydrochloride (4e)
Compound 4e was prepared from 7 and (4-acetamidophenyl)
boronic acid in 89% yield as a colorless solid, using a similar
approach to that described for 4aꢀHCl. 1H NMR (DMSO-d6): this
compound exists as a pair of rotamers at room temperature. d
2.07 (3H, s), 2.91 (minor rotamer, 3H, s), 2.96 (major rotamer,
3H, s), 3.88–4.01 (2H, m), 4.61 (minor rotamer, 2H, s), 4.63 (major
rotamer, 2H, s), 7.18–7.28 (1H, m), 7.39–7.64 (5H, m), 7.66–7.74
(2H, m), 8.22 (3H, br s), 10.19 (1H, s); MS (ESI) m/z [M+H]+ 312;
Anal. Calcd for C18H21N3O2ꢀ1.1HClꢀ1.5H2O: C, 57.12; H, 6.68; N,
11.10; Cl, 10.30. Found: C, 57.17; H, 6.68; N, 11.26; Cl, 10.41.
5.1.4. N-[(40-Chlorobiphenyl-3-yl)methyl]-N-methylglycinamide
hydrochloride (4b)
Compound 4b was prepared from 7 and (4-chlorophenyl)boro-
nic acid in 75% yield as a colorless solid, using a similar approach to
that described for 4aꢀHCl. 1H NMR (DMSO-d6): this compound
exists as a pair of rotamers at room temperature. d 2.90 (minor
rotamer, 3H, s), 2.97 (major rotamer, 3H, s), 3.90 (minor rotamer,
2H, s), 3.96 (major rotamer, 2H, s), 4.63 (minor rotamer, 2H, s),
4.64 (major rotamer, 2H, s), 7.24–7.34 (1H, m), 7.44–7.64 (5H,
m), 7.67–7.80 (2H, m), 8.30 (3H, br s); MS (FAB) m/z [M+H]+ 289;
Anal. Calcd for C16H17ClN2OꢀHCl: C, 59.09; H, 5.58; N, 8.61; Cl,
21.80. Found: C, 59.08; H, 5.61; N, 8.61; Cl, 21.75.
5.1.8. N-{[40-(Dimethylamino)biphenyl-3-yl]methyl}-N-
methylglycinamide ethanedioate (4f)
Compound 4f was prepared from 7 and [4-(dimethylamino)
phenyl]boronic acid in 75% yield as a colorless solid, using a similar
approach to that described for 4c. 1H NMR (DMSO-d6): this com-
pound exists as a pair of rotamers at room temperature. d 2.89
(minor rotamer, 3H, s), 2.93 (major rotamer, 3H, s), 2.94 (6H, s),
3.73 (minor rotamer, 2H, s), 3.79 (major rotamer, 2H, s), 4.57
(minor rotamer, 2H, s), 4.59 (major rotamer, 2H, s), 6.77–6.83
(2H, m), 7.07–7.15 (1H, m), 7.34–7.58 (5H, m); MS (ESI) m/z [M
+H]+ 298; HRMS (ESI) m/z Calcd for C18H24N3O [M+H]+:
298.1914. Found: 298.1916.
5.1.5. N-[(30-Chlorobiphenyl-3-yl)methyl]-N-methylglycinamide
ethanedioate (4c)
To a mixture of 7 (293 mg, 0.82 mmol) in toluene (4.0 mL)/
water (2.0 mL) were added (3-chlorophenyl)boronic acid
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S. Yamaki et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
5.1.9. N-Methyl-N-{[40-(morpholin-4-yl)biphenyl-3-yl]methyl}
glycinamide ethanedioate (4g)
5.1.13. tert-Butyl (2-{[(40-formylbiphenyl-3-yl)methyl](methyl)
amino}-2-oxoethyl)carbamate (8c)
Compound 4g was prepared from 7 and 4-[4-(4,4,5,5-tetram-
ethyl-1,3,2-dioxaborolan-2-yl)phenyl]morpholine in 72% yield as
a colorless solid, using a similar approach to that described for
4c. 1H NMR (DMSO-d6): this compound exists as a pair of rotamers
at room temperature. d 2.90 (minor rotamer, 3H, s), 2.95 (major
rotamer, 3H, s), 3.10–3.21 (4H, m), 3.70–3.82 (4H, m), 3.93 (minor
rotamer, 2H, s), 3.96 (major rotamer, 2H, s), 4.59 (minor rotamer,
2H, s), 4.61 (major rotamer, 2H, s), 6.99–7.06 (2H, m), 7.10–7.24
(1H, m), 7.36–7.60 (5H, m), 7.92 (2H, br s); MS (ESI) m/z [M+H]+
340; Anal. Calcd for C20H25N3O2ꢀC2H2O4: C, 61.53; H, 6.34; N,
9.78. Found: C, 61.28; H, 6.35; N, 9.75.
Compound 8c was prepared from 7 and (4-formylphenyl)boro-
nic acid in 89% yield as a beige solid, using a similar approach to
that described for 8a. 1H NMR (DMSO-d6): this compound exists
as a pair of rotamers at room temperature. d 1.38 (minor rotamer,
9H, s), 1.39 (major rotamer, 9H, s), 2.84 (minor rotamer, 3H, s),
2.95 (major rotamer, 3H, s), 3.87 (2H, d, J = 5.8 Hz), 4.60 (major
rotamer, 2H, s), 4.64 (minor rotamer, 2H, s), 6.83 (major rotamer,
1H, dd, J = 5.8, 5.8 Hz), 6.93 (minor rotamer, 1H, dd, J = 5.8,
5.8 Hz), 7.26–7.34 (1H, m), 7.48 (major rotamer, 1H, dd, J = 7.6,
7.6 Hz), 7.54 (minor rotamer, 1H, dd, J = 7.6, 7.6 Hz), 7.60–7.73
(2H, m), 7.89–8.03 (4H, m), 10.06 (1H, s); MS (FAB) m/z [M+H]+
383.
5.1.10. N-Methyl-N-{[40-(4-methylpiperazin-1-yl)biphenyl-3-yl]
methyl}glycinamide (2R,3R)-tartrate (4h)
5.1.14. 30-({[N-(tert-Butoxycarbonyl)glycyl](methyl)amino}
methyl)biphenyl-4-carboxylic acid (9)
Compound 4h was prepared from 7 and 1-methyl-4-[4-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]piperazine in 68%
yield as a colorless solid, using a similar approach to that described
for 4c. 1H NMR (DMSO-d6): this compound exists as a pair of rota-
mers at room temperature. d 2.25 (3H, s), 2.46–2.52 (4H, m), 2.90
(minor rotamer, 3H, s), 2.94 (major rotamer, 3H, s), 3.15–3.23
(4H, m), 3.86 (2H, tartaric acid), 3.87 (minor rotamer, 2H, s), 3.92
(major rotamer, 2H, s), 4.58 (minor rotamer, 2H, s), 4.61 (major
rotamer, 2H, s), 6.99–7.06 (2H, m), 7.13–7.18 (1H, m), 7.36–7.58
(5H, m); MS (ESI) m/z [M+H]+ 353; Anal. Calcd for C21H28N4OꢀC4H6-
O6ꢀ1.7H2O: C, 56.32; H, 7.07; N, 10.51. Found: C, 56.37; H, 7.18; N,
10.47.
To a solution of 8a (1.96 g, 4.76 mmol) in MeOH (20 mL) was
added 1 M NaOH aqueous solution (14.3 mL, 14.3 mmol). After
being stirred at room temperature overnight, 1 M HCl aqueous
solution (14.3 mL, 14.3 mmol) was added and the mixture was
concentrated in vacuo. The residue was diluted with water and
the mixture was extracted with CHCl3. The organic layer was dried
over MgSO4, and concentrated in vacuo to give the product (1.93 g,
quantitative yield) as a yellow solid. 1H NMR (DMSO-d6): this com-
pound exists as a pair of rotamers at room temperature. d 1.38
(minor rotamer, 9H, s), 1.40 (major rotamer, 9H, s), 2.83 (minor
rotamer, 3H, s), 2.95 (major rotamer, 3H, s), 3.87 (2H, d,
J = 5.7 Hz), 4.59 (major rotamer, 2H, s), 4.64 (minor rotamer, 2H,
s), 6.84 (major rotamer, 1H, dd, J = 5.8, 5.8 Hz), 6.93 (minor rota-
mer, 1H, dd, J = 5.8, 5.8 Hz), 7.24–7.33 (1H, m), 7.46 (major rota-
mer, 1H, dd, J = 7.6, 7.6 Hz), 7.52 (minor rotamer, 1H, dd, J = 7.6,
7.6 Hz), 7.55–7.70 (2H, m), 7.80 (major rotamer, 2H, d, J = 8.0 Hz),
7.87 (minor rotamer, 2H, d, J = 8.0 Hz), 8.02 (2H, d, J = 8.1 Hz),
13.0 (1H, br s); MS (ESI) m/z [M+H]+ 399.
5.1.11. Methyl 30-({[N-(tert-Butoxycarbonyl)glycyl](methyl)
amino}methyl)biphenyl-4-carboxylate (8a)
To a mixture of 7 (2.00 g, 5.60 mmol) in DME (30 mL)/EtOH
(5.0 mL)/water (10 mL) were added [4-(methoxycarbonyl)phe-
nyl]boronic acid (1.51 g, 8.40 mmol), Na2CO3 (1.78 g, 16.8 mmol),
and Pd(PPh3)4 (194 mg, 0.168 mmol). The mixture was stirred at
80 °C overnight. After being cooled to room temperature, the
mixture was diluted with water and extracted with CHCl3. The
organic layer was dried over MgSO4 and concentrated in vacuo.
The residue was purified by column chromatography on silica
gel (hexane/EtOAc = 1:0 to 1:2) to give the product (1.99 g, 86%)
as a yellow solid. 1H NMR (DMSO-d6): this compound exists as
a pair of rotamers at room temperature. d 1.37 (minor rotamer,
9H, s), 1.39 (major rotamer, 9H, s), 2.83 (minor rotamer, 3H, s),
2.94 (major rotamer, 3H, s), 3.81–3.95 (5H, m), 4.59 (major rota-
mer, 2H, s), 4.64 (minor rotamer, 2H, s), 6.83 (major rotamer, 1H,
dd, J = 5.7, 5.7 Hz), 6.92 (minor rotamer, 1H, dd, J = 5.8, 5.8 Hz),
7.25–7.35 (1H, m), 7.47 (major rotamer, 1H, dd, J = 7.7, 7.7 Hz),
7.52 (minor rotamer, 1H, dd, J = 7.7, 7.7 Hz), 7.57–7.71
(2H, m), 7.83 (major rotamer, 2H, d, J = 8.4 Hz), 7.89 (minor rota-
mer, 2H, d, J = 8.4 Hz), 8.04 (2H, d, J = 8.4 Hz); MS (ESI) m/z [M
+H]+ 413.
5.1.15. 30-{[Glycyl(methyl)amino]methyl}biphenyl-4-carboxylic
acid hydrochloride (10)
The mixture of 8a (167 mg, 0.40 mmol) and 6 M HCl aqueous
solution (5.00 mL, 30.0 mmol) was stirred at 100 °C for 2 h. After
being cooled to room temperature, the mixture was concentrated
in vacuo. To the residue were added EtOH and water, and the
resulting precipitate was filtered to give the product (107 mg,
79%) as a colorless solid. 1H NMR (DMSO-d6): this compound exists
as a pair of rotamers at room temperature. d 2.91 (minor rotamer,
3H, s), 2.97 (major rotamer, 3H, s), 3.92 (minor rotamer, 2H, s), 3.97
(major rotamer, 2H, s), 4.63 (minor rotamer, 2H, s), 4.65 (major
rotamer, 2H, s), 7.29–7.35 (1H, m), 7.49 (major rotamer, 1H, dd,
J = 7.7, 7.7 Hz), 7.54 (minor rotamer, 1H, dd, J = 7.7, 7.7 Hz),
7.56–7.72 (2H, m), 7.76–7.84 (2H, m), 8.01–8.06 (2H, m), 8.12
(3H, br s); MS (ESI) m/z [M+H]+ 299; Anal. Calcd for C17H18N2O3ꢀ
HClꢀ0.3H2O: C, 60.02; H, 5.81; N, 8.23; Cl, 10.42. Found: C, 59.98;
H, 5.75; N, 8.26; Cl, 10.48.
5.1.12. tert-Butyl (2-{[(40-aminobiphenyl-3-yl)methyl](methyl)
amino}-2-oxoethyl)carbamate (8b)
Compound 8b was prepared from 7 and 4-(4,4,5,5-tetramethyl-
1,3,2-dioxaborolan-2-yl)aniline in 84% yield as a pale brown solid,
using a similar approach to that described for 8a. 1H NMR (DMSO-
d6): this compound exists as a pair of rotamers at room tempera-
ture. d 1.38 (minor rotamer, 9H, s), 1.40 (major rotamer, 9H, s),
2.82 (major rotamer, 3H, s), 2.91 (minor rotamer, 3H, s), 3.85
(2H, d, J = 5.7 Hz), 4.53 (major rotamer, 2H, s), 4.57 (minor rotamer,
2H, s), 5.22 (2H, br s), 6.63 (2H, d, J = 8.4 Hz), 6.80 (major rotamer,
1H, dd, J = 5.7, 5.7 Hz), 6.87 (minor rotamer, 1H, dd, J = 5.8, 5.8 Hz),
7.05 (1H, d, J = 7.3 Hz), 7.28–7.50 (5H, m); MS (ESI) m/z [M+H]+
370.
5.1.16. 30-{[Glycyl(methyl)amino]methyl}biphenyl-4-
carboxamide hydrochloride (11a)
Compound 11a was prepared from 9 and ammonium hydroxide
in 48% yield as a colorless solid, using a similar approach to that
described for 3. 1H NMR (DMSO-d6): this compound exists as a pair
of rotamers at room temperature. d 2.91 (minor rotamer, 3H, s),
2.97 (major rotamer, 3H, s), 3.92 (minor rotamer, 2H, s), 3.97
(major rotamer, 2H, s), 4.63 (minor rotamer, 2H, s), 4.65 (major
rotamer, 2H, s), 7.27–7.34 (1H, m), 7.41 (1H, br s), 7.47 (major rota-
mer, 1H, dd, J = 7.7, 7.7 Hz), 7.53 (minor rotamer, 1H, dd, J = 7.7,
7.7 Hz), 7.55–7.71 (2H, m), 7.72–7.79 (2H, m), 7.98 (major rotamer,
Please cite this article in press as: Yamaki, S.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.10.025

S. Yamaki et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
11
2H, d, J = 8.5 Hz), 7.99 (minor rotamer, 2H, d, J = 8.4 Hz), 8.06
5.1.20. N-({40-[(Dimethylcarbamoyl)amino]biphenyl-3-yl}
methyl)-N-methylglycinamide ethanedioate (12a)
(1H, br s), 8.17 (3H, br s); MS (ESI) m/z [M+H]+ 298; Anal. Calcd
for C17H19N3O2ꢀHCl: C, 61.17; H, 6.04; N, 12.59; Cl, 10.62. Found:
C, 60.97; H, 6.08; N, 12.50; Cl, 10.59.
To a mixture of 8b (200 mg, 0.54 mmol) in CH2Cl2 (6.0 mL) were
added pyridine (66 L, 0.81 mmol) and dimethylcarbamic chloride
(55 L, 0.60 mmol). After being stirred at room temperature for
l
l
5.1.17. N-Ethyl-30-{[glycyl(methyl)amino]methyl}biphenyl-4-
carboxamide hydrochloride (11b)
1 h, the mixture was diluted with water and extracted with CHCl3.
The organic layer was dried over MgSO4, and concentrated in
vacuo. The residue was purified by column chromatography on
silica gel (CHCl3/MeOH = 98/2) to afford tert-butyl {2-[({40-
[(dimethylcarbamoyl)amino]biphenyl-3-yl}methyl)(methyl)amino]-
2-oxoethyl}carbamate (220 mg, 92%) as a colorless oil. To a
solution of tert-butyl {2-[({40-[(dimethylcarbamoyl)amino]biphe-
nyl-3-yl}methyl)(methyl)amino]-2-oxoethyl}carbamate (210 mg,
Compound 11b was prepared from 9 and ethylamine in 44%
yield as a colorless solid, using a similar approach to that described
for 3. 1H NMR (DMSO-d6): this compound exists as a pair of rota-
mers at room temperature. d 1.14 (3H, t, J = 7.2 Hz), 2.91 (minor
rotamer, 3H, s), 2.97 (major rotamer, 3H, s), 3.27–3.32 (2H, m),
3.92 (minor rotamer, 2H, s), 3.97 (major rotamer, 2H, s), 4.63
(minor rotamer, 2H, s), 4.65 (major rotamer, 2H, s), 7.27–7.33
(1H, m), 7.47 (major rotamer, 1H, dd, J = 7.7, 7.7 Hz), 7.53 (minor
rotamer, 1H, dd, J = 7.7, 7.7 Hz), 7.55–7.70 (2H, m), 7.72–7.80
(2H, m), 7.95 (major rotamer, 2H, d, J = 8.4 Hz), 7.96 (minor rota-
mer, 2H, d, J = 8.4 Hz), 8.16 (3H, br s), 8.56 (1H, t, J = 5.4 Hz); MS
(ESI) m/z [M+H]+ 326; Anal. Calcd for C19H23N3O2ꢀHCl: C, 63.06;
H, 6.68; N, 11.61; Cl, 9.80. Found: C, 62.96; H, 6.70; N, 11.57; Cl,
9.81.
0.48 mmol) in MeOH was added
4 M HCl/EtOAc (1.20 mL,
4.80 mmol). After being stirred at room temperature for 4 h, the
mixture was concentrated in vacuo. The residue was diluted with
saturated NaHCO3 aqueous solution, and the mixture was
extracted with CHCl3. The organic layer was dried over MgSO4
and concentrated in vacuo. To a solution of the residue in EtOH
was added oxalic acid (43 mg, 0.48 mmol). The resulting precipi-
tate was filtered to give the product (125 mg, 61%) as a colorless
solid. 1H NMR (DMSO-d6): this compound exists as a pair of rota-
mers at room temperature. d 2.90 (minor rotamer, 3H, s), 2.94
(major rotamer, 3H, s), 2.94 (6H, s), 3.85 (minor rotamer, 2H, s),
3.89 (major rotamer, 2H, s), 4.59 (minor rotamer, 2H, s), 4.61
(major rotamer, 2H, s), 7.15–7.22 (1H, m), 7.33–7.48 (2H, m),
7.48–7.61 (5H, m), 8.43 (1H, br s); MS (ESI) m/z [M+H]+ 341; HRMS
5.1.18. N,N-Diethyl-30-{[glycyl(methyl)amino]methyl}biphenyl-
4-carboxamide ethanedioate (11c)
To a solution of 9 (210 mg, 0.53 mmol) in DMF (5.0 mL) were
added N-ethylethanamine (42 mg, 0.58 mmol), WSCD (90 mg,
0.58 mmol), HOBt (78 mg, 0.58 mmol) and Et3N (80 lL, 0.58 mmol)
at room temperature. After being stirred at the same temperature
overnight, the mixture was concentrated in vacuo. The residue was
diluted with water and the mixture was extracted with EtOAc. The
organic layer was dried over MgSO4, and concentrated in vacuo.
The residue was purified by column chromatography on silica gel
(hexane/EtOAc = 1:0 to 3:7) to give tert-butyl {2-[{[40-(diethylcar-
bamoyl)biphenyl-3-yl]methyl}(methyl)amino]-2-oxoethyl}carba-
(ESI) m/z Calcd for C
19H25N4O2 [M+H]+: 341.1972. Found:
341.1974.
5.1.21. N-(30-{[Glycyl(methyl)amino]methyl}biphenyl-4-
yl)morpholine-4-carboxamide ethanedioate (12b)
Compound 12b was prepared from 8b and morpholine-4-car-
bonyl chloride and Et3N instead of pyridine in 57% yield as a solid,
using a similar approach to that described for 12a. 1H NMR (DMSO-
d6): this compound exists as a pair of rotamers at room tempera-
ture. d 2.91 (minor rotamer, 3H, s), 2.95 (major rotamer, 3H, s),
3.40–3.49 (4H, m), 3.57–3.66 (4H, m), 3.92 (minor rotamer, 2H,
s), 3.96 (major rotamer, 2H, s), 4.60 (minor rotamer, 2H, s), 4.62
(major rotamer, 2H, s), 7.17–7.23 (1H, m), 7.38–7.60 (7H, m),
8.67 (major rotamer, 1H, s), 8.68 (minor rotamer, 1H, s); MS (ESI)
m/z [M+H]+ 383; Anal. Calcd for C21H26N4O3ꢀC2H2O4ꢀ0.7H2O: C,
56.95; H, 6.11; N, 11.55. Found: C, 56.88; H, 6.14; N, 11.57.
mate. To
a
solution of tert-butyl {2-[{[40-(diethylcarbamoyl)
biphenyl-3-yl]methyl}(methyl)amino]-2-oxoethyl}carbamate in
EtOH (10 mL) was added 4 M HCl/EtOAc (10.0 mL, 40.0 mmol).
After being stirred at room temperature for 2 h, the mixture was
concentrated in vacuo. The residue was diluted with saturated
NaHCO3 aqueous solution, and the mixture was extracted with
CHCl3. The organic layer was dried over Na2SO4, and concentrated
in vacuo. To a solution of the residue in EtOH was added oxalic acid
(47 mg, 0.53 mmol). The mixture was concentrated in vacuo. To
the residue were added EtOH and water, and the resulting precip-
itate was filtered to give the product (90 mg, 39%) as a colorless
solid. 1H NMR (DMSO-d6): this compound exists as a pair of rota-
mers at room temperature. d 1.00–1.24 (6H, br m), 2.91 (minor
rotamer, 3H, s), 2.96 (major rotamer, 3H, s), 3.12–3.55 (4H, br
m), 3.94 (minor rotamer, 2H, s), 3.97 (major rotamer, 2H, s), 4.62
(minor rotamer, 2H, s), 4.65 (major rotamer, 2H, s), 7.27–7.33
(1H, m), 7.40–7.53 (3H, m), 7.53–7.69 (2H, m), 7.69–7.78 (2H,
m); MS (FAB) m/z [M+H]+ 354; Anal. Calcd for C21H27N3O2ꢀC2H2O4:
C, 62.29; H, 6.59; N, 9.47. Found: C, 62.03; H, 6.55; N, 9.54.
5.1.22. N-({40-[(Diethylamino)methyl]biphenyl-3-yl}methyl)-N-
methylglycinamide dihydrochloride (13)
To a solution of 8c (250 mg, 0.65 mmol) in DCE (5.0 mL) were
added diethylamine (205 lL, 1.96 mmol) and AcOH (187 lL,
3.27 mmol). After being stirred at room temperature for 1 h,
sodium triacetoxyborohydride (416 mg, 1.96 mmol) was added.
After being stirred at room temperature for 1 h, the mixture was
diluted with saturated NaHCO3 aqueous solution and extracted
with CHCl3. The organic layer was concentrated in vacuo. The resi-
due was purified by column chromatography on amino silica gel
(hexane/EtOAc = 1:0 to 1:4) to afford tert-butyl {2-[({40-[(diethy-
lamino)methyl]biphenyl-3-yl}methyl)(methyl)amino]-2-oxoethyl}
carbamate. To a solution of tert-butyl {2-[({40-[(diethylamino)
methyl]biphenyl-3-yl}methyl)(methyl)amino]-2-oxoethyl}carba-
mate in EtOAc was added 4 M HCl/EtOAc (10.0 mL, 40.0 mmol).
After being stirred at room temperature for 2 h, the mixture was
concentrated in vacuo and the residue was diluted with EtOH/
EtOAc. The resulting precipitate was filtered to give the product
(177 mg, 66%) as a light pink solid. 1H NMR (DMSO-d6): this com-
pound exists as a pair of rotamers at room temperature. d 1.27 (6H,
t, J = 7.2 Hz), 2.91 (minor rotamer, 3H, s), 2.97 (major rotamer, 3H,
s), 2.99–3.14 (4H, m), 3.89–4.00 (2H, m), 4.32 (2H, d, J = 5.6 Hz),
5.1.19. N-Methyl-N-{[40-(morpholin-4-ylcarbonyl)biphenyl-3-
yl]methyl}glycinamide (2R,3R)-tartrate (11d)
Compound 11d was prepared from 9 and morpholine in 71%
yield as a colorless solid, using a similar approach to that described
for 11c. 1H NMR (DMSO-d6): this compound exists as a pair of rota-
mers at room temperature. d 2.89 (minor rotamer, 3H, s), 2.93
(major rotamer, 3H, s), 3.31–3.71 (8H, m), 3.68 (minor rotamer,
2H, s), 3.74 (major rotamer, 2H, s), 3.77 (2H, s, tartaric acid), 4.60
(minor rotamer, 2H, s), 4.63 (major rotamer, 2H, s), 7.23–7.32
(1H, m), 7.44–7.68 (5H, m), 7.69–7.76 (2H, m); MS (ESI) m/z [M
+H]+ 368; HRMS (ESI) m/z Calcd for C21H26N3O [M+H]+:
368.1969. Found: 368.1971.
Please cite this article in press as: Yamaki, S.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.10.025

12
S. Yamaki et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
4.63 (minor rotamer, 2H, s), 4.64 (major rotamer, 2H, s), 7.26–7.33
(1H, m), 7.47 (major rotamer, 1H, dd, J = 7.6, 7.6 Hz), 7.52 (minor
rotamer, 1H, dd, J = 7.6, 7.6 Hz), 7.53 (minor rotamer, 1H, s), 7.58
(major rotamer, 1H, s), 7.62 (major rotamer, 1H, d, J = 7.6 Hz),
7.67 (minor rotamer, 1H, d, J = 7.6 Hz), 7.71–7.80 (4H, m), 8.11–
8.29 (3H, m), 10.92 (1H, br s); MS (FAB) m/z [M+H]+ 340. Anal.
Calcd for C21H29N3Oꢀ2.1HClꢀ1.8H2O: C, 56.24; H, 7.80; N, 9.37; Cl,
16.60. Found: C, 56.48; H, 7.95; N, 9.13; Cl, 16.87.
71%) as a solid: 1H NMR (DMSO-d6): this compound exists as a pair
of rotamers at room temperature. d 2.43 (minor rotamer, 6H, s),
2.50 (major rotamer, 6H, s), 2.81 (minor rotamer, 3H, s), 2.96
(major rotamer, 3H, s), 3.13–3.19 (4H, m), 3.58 (minor rotamer,
2H, s), 3.69 (major rotamer, 2H, s), 3.73–3.78 (4H, m), 4.11 (2H,
s, tartaric acid), 4.58 (major rotamer, 2H, s), 4.65 (minor rotamer,
2H, s), 7.00–7.06 (2H, m), 7.11–7.17 (1H, m), 7.35–7.47 (2H, m),
7.48–7.57 (3H, m); MS (ESI) m/z [M+H]+ 368; Anal. Calcd for C22
-
H
29N3O2ꢀC4H6O6ꢀ2H2O: C, 56.41; H, 7.10; N, 7.59. Found: C,
5.1.23. N-Methyl-1-[40-(morpholin-4-yl)biphenyl-3-yl]
methanamine (16)
56.65; H, 6.92; N, 7.25.
To a mixture of tert-butyl (3-bromobenzyl)methylcarbamate
(14; 2.10 g, 7.00 mmol) in DME (21 mL)/water(11 mL) were added
4-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]morpho-
line (15; 2.12 g, 7.35 mmol), Pd(PPh3)4 (243 mg, 0.21 mmol) and
Na2CO3 (2.22 g, 21.0 mmol). The mixture was stirred at 90 °C for
36 h. After being cooled to room temperature, the mixture was
concentrated in vacuo. The residue was diluted with water and
extracted with CHCl3. The organic layer was dried over MgSO4,
and concentrated in vacuo. The residue was purified by column
chromatography on silica gel (CHCl3/MeOH = 98/2) to afford
5.1.26. N-Methyl-N-{[40-(morpholin-4-yl)biphenyl-3-yl]
methyl}-b-alaninamide (2R,3R)-tartrate (19)
Compound 19 was prepared from 16 and N-(tert-butoxycar-
bonyl)-b-alanine in 69% yield as a solid, using a similar approach
to that described for 11c. 1H NMR (DMSO-d6): this compound
exists as a pair of rotamers at room temperature. d 2.76 (2H, t,
J = 6.4 Hz), 2.89 (minor rotamer, 3H, s), 2.94 (major rotamer, 3H,
s), 2.99–3.24 (2H, m), 3.10–3.20 (4H, m), 3.70–3.79 (4H, m), 3.83
(2H, s, tartaric acid), 4.59 (major rotamer, 2H, s), 4.62 (minor rota-
mer, 2H, s), 6.97–7.07 (2H, m), 7.10–7.21 (1H, m), 7.34–7.59 (5H,
m); MS (ESI) m/z [M+H]+ 354; Anal. Calcd for C21H27N3O2ꢀC4H6O6-
ꢀ0.7H2O: C, 58.17; H, 6.72; N, 8.14. Found: C, 58.02; H, 6.60; N, 8.20.
tert-butyl
carbamate (2.20 g, 82%) as a colorless oil. To a solution of tert-
butyl
methyl{[40-(morpholin-4-yl)biphenyl-3-yl]methyl}carba-
methyl{[40-(morpholin-4-yl)biphenyl-3-yl]methyl}
mate (1.82 g, 4.76 mmol) in EtOH (20 mL) was added 4 M HCl/
EtOAc (10.0 mL, 40.0 mmol). After being stirred at room tempera-
ture for 24 h, the mixture was concentrated in vacuo. The residue
was diluted with saturated NaHCO3 aqueous solution, and the mix-
ture was extracted with CHCl3. The organic layer was dried over
Na2SO4 and concentrated in vacuo to afford the product (1.24 g,
92%). 1H NMR (CDCl3): d 2.49 (3H, s), 3.17–3.25 (4H, m), 3.81
(2H, s), 3.86–3.91 (4H, m), 6.95–7.01 (2H, m), 7.25 (1H, d,
J = 7.7 Hz), 7.37 (1H, dd, J = 7.6, 7.6 Hz), 7.44–7.48 (1H, m), 7.51–
7.57 (3H, m); MS (ESI) m/z [M+H]+ 283.
5.1.27. N,2-Dimethyl-N-{[40-(morpholin-4-yl)biphenyl-3-yl]
methyl}alaninamide (2R,3R)-tartrate (27)
To a mixture of 6 (500 mg, 2.50 mmol) in CH2Cl2 (5.0 mL) were
added
N-(tert-butoxycarbonyl)-2-methylalanine
(533 mg,
2.62 mmol), WSCDꢀHCl (527 mg, 2.75 mmol), and HOBt (371 mg,
2.75 mmol). After being stirred at room temperature for 3 h, the
mixture was diluted with water and extracted with CHCl3. The
organic layer was washed with HCl aqueous solution and saturated
NaHCO3 aqueous solution, dried over MgSO4, and concentrated in
vacuo. The residue was purified by column chromatography on sil-
ica gel (CHCl3/MeOH = 98:2) to afford tert-butyl {1-[(3-bromoben-
zyl)(methyl)amino]-2-methyl-1-oxopropan-2-yl}carbamate (23)
(915 mg, 95%) as a colorless oil. To a mixture of 23 (286 mg,
0.74 mmol) in DME (2.9 mL)/water (1.4 mL) were added 15
(225 mg, 0.78 mmol), Na2CO3 (236 mg, 2.23 mmol), and Pd
(PPh3)4 (26 mg, 0.022 mmol). The mixture was stirred at 90 °C for
36 h. After being cooled to room temperature, the mixture was
concentrated in vacuo. The residue was diluted with water and
extracted with CHCl3. The organic layer was dried over MgSO4
and concentrated in vacuo. The residue was purified by column
chromatography on silica gel (CHCl3/MeOH = 98:2) to afford tert-
5.1.24. N,N2-Dimethyl-N-{[40-(morpholin-4-yl)biphenyl-3-yl]
methyl}glycinamide (2R,3R)-tartrate (17)
Compound 17 was prepared from 16 and N-(tert-butoxycar-
bonyl)-N-methylglycine in 43% yield, using a similar approach to
that described for 11c. 1H NMR (DMSO-d6): this compound exists
as a pair of rotamers at room temperature. d 2.57 (minor rotamer,
3H, s), 2.59 (major rotamer, 3H, s), 2.88 (minor rotamer, 3H, s), 2.93
(major rotamer, 3H, s), 3.10–3.21 (4H, m), 3.72–3.80 (4H, m), 4.11
(minor rotamer, 2H, s), 4.13 (major rotamer, 2H, s), 4.13 (2H, s, tar-
taric acid), 4.56 (minor rotamer, 2H, s), 4.61 (major rotamer, 2H, s),
7.00–7.07 (2H, m), 7.14–7.20 (1H, m), 7.37–7.59 (5H, m); MS (ESI)
m/z [M+H]+ 354; HRMS (ESI) m/z Calcd for C21H28N3O2 [M+H]+:
354.2176. Found: 354.2175.
butyl
[2-methyl-1-(methyl{[40-(morpholin-4-yl)biphenyl-3-yl]
methyl}amino)-1-oxopropan-2-yl]carbamate (290 mg, 84%) as a
colorless oil. To a solution of tert-butyl [2-methyl-1-(methyl{[40-(-
morpholin-4-yl)biphenyl-3-yl]methyl}amino)-1-oxopropan-2-yl]-
carbamate (290 mg, 0.62 mmol) in MeOH was added 4 M HCl/
EtOAc. After being stirred at room temperature for 4 h, the mixture
was concentrated in vacuo. The residue was diluted with saturated
NaHCO3 aqueous solution, and the mixture was extracted with
CHCl3. The organic layer was dried over Na2SO4 and concentrated
in vacuo. To a solution of the residue in EtOH was added (2R,3R)-
tartaric acid (93 mg, 0.62 mmol). The resulting precipitate was fil-
tered to give the product (160 mg, 50%) as a colorless solid. 1H
NMR (DMSO-d6): this compound exists as a pair of rotamers at
room temperature. d 1.55 (minor rotamer, 6H, s), 1.58 (major rota-
mer, 6H, s), 2.95–3.21 (7H, m), 3.73–3.78 (4H, m), 3.86 (2H, s, tar-
taric acid), 4.68 (2H, br s), 6.98 (minor rotamer, 2H, d, J = 8.9 Hz),
7.03 (major rotamer, 2H, d, J = 8.9 Hz), 7.12 (major rotamer, 1H,
d, J = 7.5 Hz), 7.22 (minor rotamer, 1H, d, J = 7.5 Hz), 7.31–7.44
(2H, m), 7.46–7.57 (3H, m); MS (ESI) m/z [M+H]+ 368; Anal. Calcd
5.1.25. N,N2,N2-Trimethyl-N-{[40-(morpholin-4-yl)biphenyl-3-
yl]methyl}glycinamide (2R,3R)-tartrate (18)
To a solution of 16 (200 mg, 0.71 mmol) in DCE (3.0 mL) were
added N,N-dimethylglycine (80 mg, 0.78 mmol), WSCDꢀHCl
(163 mg, 0.85 mmol), and HOBt (115 mg, 0.85 mmol). After being
stirred at room temperature for 2 h, the mixture was diluted with
saturated NaHCO3 aqueous solution and extracted with CHCl3. The
organic layer was dried over Na2SO4, and concentrated in vacuo.
The residue was purified by column chromatography on silica gel
(CHCl3/MeOH) to afford N,N2,N2-trimethyl-N-{[40-(morpholin-4-
yl)biphenyl-3-yl]methyl}glycinamide. To a solution of N,N2,N2-tri-
methyl-N-{[40-(morpholin-4-yl)biphenyl-3-yl]methyl}glycinamide
in EtOH (3.0 mL) was added (2R,3R)-tartaric acid (106 mg,
0.71 mmol). After being stirred at room temperature for 2 h, the
mixture was concentrated in vacuo to give the product (261 mg,
Please cite this article in press as: Yamaki, S.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.10.025

S. Yamaki et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
13
for C22H29N3O2ꢀC4H6O6ꢀH2O: C, 58.31; H, 6.96; N, 7.85. Found: C,
5.1.31. 2-Amino-1-{5-[4-(morpholin-4-yl)phenyl]-1,3-dihydro-
2H-isoindol-2-yl}ethanone hydrochloride (30)
58.57; H, 6.78; N, 7.83.
Compound 30 was prepared from 26 and 15 in 85% yield as a
solid, using a similar approach to that described for 4aꢀHCl. 1H
NMR (DMSO-d6): d 3.21–3.35 (4H, m), 3.78–3.98 (6H, m), 4.75
(2H, d, J = 8.6 Hz), 4.91 (2H, d, J = 8.1 Hz), 7.25–7.33 (2H, m), 7.43
(1H, dd, J = 8.1, 10.8 Hz), 7.57–7.69 (4H, m), 8.36 (3H, br s); MS
(ESI) m/z [M+H]+ 338; HRMS (ESI) m/z Calcd for C20H24N3O2 [M
+H]+: 338.1863. Found:338.1865.
5.1.28. N-Isopropyl-N-{[40-(morpholin-4-yl)biphenyl-3-yl]
methyl}glycinamide (2R,3R)-tartrate (28)
Compound 28 was prepared from N-(3-bromobenzyl)propan-2-
amine (20) and [(tert-butoxycarbonyl)amino]acetic acid in 72%
yield, using a similar approach to that described for 27. 1H NMR
(DMSO-d6): this compound exists as a pair of rotamers at room
temperature. d 1.05–1.18 (6H, m), 3.08–3.22 (4H, m), 3.66 (minor
rotamer, 2H, s), 3.71–3.79 (4H, m), 3.85 (2H, s, tartaric acid), 4.03
(major rotamer, 2H, s), 4.48–4.63 (3H, m), 6.97–7.08 (2H, m),
7.11–7.20 (1H, m), 7.29–7.58 (5H, m); MS (ESI) m/z [M+H]+ 368;
Anal. Calcd for C22H29N3O2ꢀC4H6O6ꢀ0.6H2O: C, 59.10; H, 6.91; N,
7.95. Found: C, 58.97; H, 6.76; N, 7.95.
5.1.32. Methyl 40-(morpholin-4-yl)biphenyl-3-carboxylate (32)
To
a solution of methyl 3-bromobenzoate (31; 500 mg,
2.33 mmol) in DME (5.0 mL)/water (2.0 mL) were added 15
(700 mg, 2.42 mmol), Na2CO3 (739 mg, 6.98 mmol), and Pd(PPh3)4
(80 mg, 0.069 mmol). The mixture was stirred at 80 °C overnight.
After being cooled to room temperature, the mixture was concen-
trated in vacuo. The residue was diluted with saturated NaHCO3
aqueous solution and extracted with CHCl3. The organic layer was
dried over Na2SO4 and concentrated in vacuo. The residue was puri-
fied by column chromatography on silica gel (CHCl3/MeOH) to
5.1.29. N-Methyl-N-{[40-(morpholin-4-yl)biphenyl-3-yl]methyl}
ethane-1,2-diamine (2R,3R)-tartrate (29)
To
a solution of 1-bromo-3-(bromomethyl)benzene (21;
570 mg, 2.28 mmol) in DMF (6.0 mL) were added tert-butyl [2-
(methylamino)ethyl]carbamate (400 mg, 2.30 mmol) and K2CO3
(630 mg, 4.56 mmol). After being stirred at room temperature
overnight, the mixture was concentrated in vacuo. The residue
was diluted with saturated NaHCO3 aqueous solution and
extracted with CHCl3. The organic layer was dried over Na2SO4,
and concentrated in vacuo. The residue was purified by column
chromatography on silica gel (CHCl3/MeOH) to afford tert-butyl
afford
methyl
40-(morpholin-4-yl)biphenyl-3-carboxylate
(531 mg, 77%). To a solution of methyl 40-(morpholin-4-yl)biphe-
nyl-3-carboxylate (531 mg, 1.79 mmol) in EtOH (10 mL) was added
1 M NaOH aqueous solution (5.00 mL, 5.00 mmol). The mixture was
stirred at 60 °C for 3 h. After being cooled to room temperature, the
mixture was neutralized with 1 M HCl aqueous solution. The result-
ing precipitate was filtered to give the product (470 mg, 93%) as a
solid. 1H NMR (DMSO-d6): d 3.12–3.21 (4H, m), 3.72–3.80 (4H,
m), 7.05 (2H, d, J = 7.8 Hz), 7.52–7.63 (3H, m), 7.83–7.89 (2H, m),
8.11–8.15 (1H, m), 13.01 (1H, br s); MS (ESI) m/z [M+H]+ 284.
{2-[(3-bromobenzyl)(methyl)amino]ethyl}carbamate
(25;
782 mg, quantitative yield). To mixture of 25 (500 mg,
a
1.46 mmol) in DME (5.0 mL)/water (2.0 mL) were added 15
(500 mg, 1.73 mmol), Na2CO3 (463 mg, 4.37 mmol), and Pd
(PPh3)4 (55 mg, 0.048 mmol). The mixture was stirred at 80 °C
overnight. After being cooled to room temperature, the mixture
was concentrated in vacuo. The residue was diluted with saturated
NaHCO3 aqueous solution and extracted with CHCl3. The organic
layer was dried over Na2SO4 and concentrated in vacuo. The resi-
due was purified by column chromatography on silica gel (CHCl3/
MeOH) to afford tert-butyl [2-(methyl{[40-(morpholin-4-yl)biphe-
nyl-3-yl]methyl}amino)ethyl]carbamate. To a solution of tert-butyl
[2-(methyl{[40-(morpholin-4-yl)biphenyl-3-yl]methyl}amino)
ethyl]carbamate in EtOH (6.0 mL) was added 4 M HCl/EtOAc
(3.00 mL, 12.0 mmol). After being stirred at room temperature
overnight, the mixture was concentrated in vacuo. The residue
was diluted with saturated NaHCO3 aqueous solution, and the mix-
ture was extracted with CHCl3. The organic layer was dried over
Na2SO4 and concentrated in vacuo. To a solution of the residue in
EtOH was added (2R,3R)-tartaric acid (104 mg, 0.69 mmol). After
being stirred at room temperature for 2 h, the mixture was concen-
trated in vacuo. To the residue was added EtOAc and the resulting
precipitate was filtered to give the product (111 mg, 16%). 1H NMR
(DMSO-d6): d 2.16 (3H, s), 2.58 (2H, t, J = 6.2 Hz), 2.95 (2H, t,
J = 6.2 Hz), 3.08–3.22 (4H, m), 3.58 (2H, s), 3.73–3.78 (4H, m),
4.02 (2H, s, tartaric acid), 7.03 (2H, d, J = 8.8 Hz), 7.26 (1H, d,
J = 7.6 Hz), 7.37 (1H, dd, J = 7.6, 7.6 Hz), 7.50 (1H, d, J = 7.8 Hz),
7.52–7.65 (3H, m); MS (ESI) m/z [M+H]+ 326; HRMS (ESI) m/z Calcd
for C20H28N3O [M+H]+: 326.2227. Found:326.2230.
5.1.33. N-(2-Aminoethyl)-N-methyl-40-(morpholin-4-yl)
biphenyl-3-carboxamide (2R,3R)-tartrate (33)
Compound 33 was prepared from 32 and tert-butyl [2-(methy-
lamino)ethyl]carbamate in 34% yield, using a similar approach to
that described for 11c. 1H NMR (DMSO-d6): this compound exists
as a pair of rotamers at room temperature. d 2.96 (3H, br s),
3.03–3.21 (6H, m), 3.63–3.79 (6H, m), 3.87 (2H, s, tartaric acid),
6.97–7.09 (2H, m), 7.22–7.41 (1H, m), 7.43–7.54 (1H, m), 7.59
(major rotamer, 2H, d, J = 8.8 Hz), 7.64 (minor rotamer, 2H, d,
J = 8.8 Hz), 7.69 (major rotamer, 2H, d, J = 7.3 Hz), 7.78 (minor rota-
mer, 2H, d, J = 7.4 Hz); MS (ESI) m/z [M+H]+ 340; Anal. Calcd for
C20H25N3O2ꢀC4H6O6ꢀ0.6H2O: C, 57.61; H, 6.49; N, 8.40. Found: C,
57.61; H, 6.42; N, 8.37.
5.2. Molecular modeling
5.2.1. Human VAP-1 model
A
side-chain conformational search and minimization for
Leu469 of the three-dimensional (3D) structure of VAP-1 with
TPQ in an active conformation (PDB-code: 2C11,18 resolution:
2.90 Å) was performed using the Low Mode MD19 function in the
Molecular Operating Environment (MOE) program16a with an
MMFF94x forcefield. The bound 2-hidrazinopyridine ligand was
then deleted.
5.2.2. Docking study
The ligand molecules were prepared using LigPrep20 and Conf-
gen,21 and the energy-minimized conformation was used to input
molecules into the follow docking calculations. Compounds were
docked to the human VAP-1 model using the docking program
GOLD version 5.2.22 The ligand-binding pocket was defined using
CbH from Leu468 as the central atom with a radius of 20 Å. The
ligand was docked covalently to nitrogen atom N1 of PAQ1729
(PAQ is used PDB entry 2C1118) to assign TPQ and the ligand. Each
ligand was docked 10 times.
5.1.30. tert-Butyl [2-(5-bromo-1,3-dihydro-2H-isoindol-2-yl)-2-
oxoethyl]carbamate (26)
Compound 26 was prepared from 5-bromoisoindoline
hydrochloride (22) and [(tert-butoxycarbonyl)amino]acetic acid
in 48% yield, using a similar approach to that described for 7. 1H
NMR (CDCl3): d 1.47 (9H, s), 4.00 (2H, d, J = 4.3 Hz), 4.74 (2H, d,
J = 14.5 Hz), 4.79 (2H, d, J = 16.4 Hz), 5.48 (1H, br s), 7.17 (1H, dd,
J = 8.1, 13.5 Hz), 7.42–7.48 (2H, m); MS (ESI) m/z [M+H]+ 357.
Please cite this article in press as: Yamaki, S.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.10.025

14
S. Yamaki et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
5.3. Inhibitory effect on human and rat VAP-1 enzyme activity
blood to prepare blood samples of 1 lg/mL, and then the blood
sample was incubated at 37 °C for up to 6 h (n = 3). After
incubation, test compound in the blood samples was extracted
by deproteination with acetonitrile, and then analyzed by LC-MS/
MS.
Human and rat VAP-1 enzyme activity was measured by a
radiochemistry-enzymatic assay using 14C-benzylamine (American
Radiolabeled Chemicals, STL, USA).23 An enzyme suspension pre-
pared from CHO (Chinese Hamster Ovary) cells expressing a
human or rat VAP-1 enzyme was preincubated with the test com-
pound in a 96-well microplate at room temperature for 20 min.
Subsequently, the enzyme suspension was incubated with 14C-
benzylamine (final concentration of 1 ꢁ 10ꢂ6 mol/L) to a final vol-
5.4.3. Rat pharmacokinetic study
Male Wistar rats were purchased from Charles River Laborato-
ries Japan, Inc. (Yokohama, Japan). Rats were fasted for approxi-
mately 17 h before dosing. Compound 2 or 4g was administered
to rats (n = 3) either intravenously (iv) or orally (po) at a dose of
1 mg/kg. For the iv study, test compound was prepared as a solu-
tion of 10% DMF, 10% propylene glycol and 80% saline. For the po
study, test compound was prepared as a solution of 10% DMF,
10% propylene glycol and 80% water. Blood was collected from
the vein using heparin as an anticoagulant, immediately chilled
on ice, and centrifuged to obtain plasma fraction. Test compound
in plasma samples were extracted by deproteination with acetoni-
trile, and then analyzed by LC-MS/MS. Pharmacokinetic parame-
ters were calculated from plasma concentrations of test
compound using the non-compartmental analysis model.
ume of 50
by the addition of 2 mol/L (50
ucts were extracted directly in a 200-
l
L at 37 °C for 1 h. The enzymatic reaction was stopped
L) of citric acid. The oxidation prod-
L toluene scintillator, and
l
l
the radioactivity was measured with a scintillation spectrometer.
5.4. Animal experiments
All animal experimental procedures were approved by the Insti-
tutional Animal Care and Use Committee of Astellas Pharma Inc.
Further, Astellas Pharma Inc. Tsukuba Research Center was
awarded Accreditation Status by the AAALAC International. All
efforts were made to minimize the number of animals used and
to avoid suffering and distress.
5.5. Permeability study using artificial membrane
5.4.1. Inhibitory effect on plasma VAP-1 enzyme activities in rat
Male Wistar rats were purchased from Japan Charles River Lab-
oratories (Yokohama, Japan). Blood samples for the measurement
of VAP-1 activity were collected via the tail vein under diethyl
ether anesthesia through a heparinized capillary. Blood was sam-
pled before and 1 h after the orally dosing of test compounds.
The collected blood was placed into sampling tubes (1.5 mL),
cooled in iced water and centrifuged at 4 °C to obtain plasma.
The resulting plasma was stored at ꢂ80 °C until measurement of
plasma VAP-1 activity.
Parallel artificial membrane permeability assays (PAMPA) con-
ducted in this study used the PAMPA Evolution from pION Inc. (Bil-
lerica, MA). In this assay, a ‘sandwich’ is formed from a 96-well
microtiter plate (pION Inc.) and a 96-well filter plate (pION Inc.),
so that each composite well is divided into two chambers, with a
donor at the bottom and an acceptor at the top, separated by a
microfilter disc coated with lipid (pION Inc.) in each well. Drug
samples were introduced as a 10 mM dimethyl sulfoxide (DMSO)
stock solution in a 96-well polypropylene microtiter plate. The
robotic liquid handling system draws a 5 lL aliquot of the DMSO
Plasma VAP-1 activity was measured using a radioenzyme
assay with a substrate of 14C-benzylamine (American Radiolabeled
Chemicals, STL, USA)..23 Total VAP-1 activity was measured by
stock solution and mixes it into an aqueous buffer solution of
10% (v/v) DMSO to attain a final typical sample concentration of
50 lM. The drug solutions were filtered using a 96-well filter plate
reacting a mixture of 80 or 100
lamine at 37 °C for 2 h, and non-specific activity was measured
with 10 aminoguanidine hydrochloride (Sigma, STL, USA,
lL rat plasma and 10
l
L 14C-benzy-
(polyvinylidene fluoride; Corning Inc., Corning, NY) and added to
the donor compartments. The donor solution was adjusted to pH
6.5 (NaOH-treated buffer; pION Inc.), whereas the acceptor solu-
tion had a pH of 7.4 (pION Inc.). The plates were sandwiched
together and incubated at room temperature for 2 h in a humid-
ity-saturated atmosphere. After incubation, the sandwiched plates
were separated, and both the donor and acceptor compartments
were assayed for the amount of material present by comparison
of the peak heights by HPLC. Mass balance, which is the difference
between the reference and the sum of the donor and acceptor, was
used to determine the amount of material remaining in the mem-
brane barrier. The permeability (Pe) was calculated using PAMPA
Evolution software (pION Inc.).
lL
1 mmol/L), FR271207 or FR299676 (a specific VAP-1 inhibitor;
Astellas Pharma Inc., 1 mmol/L) under the same conditions.
Radioactivity of the reaction metabolite (14C-benzaldehyde) was
measured with a liquid scintillation counter (TRI-CARB 2100TR or
TRI-CARB 3110TR; Perkin Elmer Japan, Yokohama, Japan). Total
and non-specific VAP-1 activities (pmol) were calculated using
the measured radioactivity and the radioactivity of a standard with
14C-benzylamine. Plasma VAP-1 activity and inhibition ratio were
calculated by the following formula:
Plasma VAP-1 activity ðpmol=mL=hÞ
¼ ½total activity ðpmolÞ ꢂ non-specific activity ðpmolÞꢃ
5.6. Plasma protein binding study
ꢁ ½1000=80 or 100 ð
l
LÞꢃ=2 ðhÞ:
Plasma was obtained from male Wistar rats purchased from
Charles River Laboratories Japan, Inc. (Yokohama, Japan). Test com-
pound solution of 4a (hydrochloride salt), 4b or 4g was added to
Inhibition ratio ð%Þ
¼ ½Plasma VAP-1 activity ðbefore dosingÞ
ꢂ Plasma VAP-1activity ð1 h after dosingÞꢃ
=Plasma VAP-1 activity ðbefore dosingÞ
the plasma to prepare plasma samples of 2 lM. The plasma sample
and PBS were transferred into the sample chamber and buffer
chamber of a RED device (Thermo Fisher Scientific Inc., USA),
respectively (n = 3). The dialysis device was placed on an orbital
shaker in a CO2 incubator set at 37 °C and 5% CO2, and incubated
for 16 h. After incubation, the plasma sample and PBS sample were
collected from the two chambers, and mixed with the same vol-
ume of blank PBS and blank plasma, respectively. Test compound
in mixed samples was extracted by deproteination with acetoni-
trile, and then analyzed by LC-MS/MS.
5.4.2. Stability in rat blood
Male Wistar rats were purchased from Charles River
Laboratories Japan, Inc. (Yokohama, Japan). Blood was collected
from the abdominal artery using heparin as an anticoagulant. Test
compound solution of 3 or 4a (hydrochloride salt) was added to the
Please cite this article in press as: Yamaki, S.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.10.025

S. Yamaki et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
15
U.; Pentikäinen, O.; Fülöp, F.; Jalkanen, S.; Salmi, M. Arthritis Rheum. 2006,
54, 2852; (b) Akin, E.; Aversa, J.; Steere, A. C. Infect. Immun. 2001,
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The plasma protein binding ratio (%) and plasma unbound frac-
tion (fp) were calculated using the following equations:
7. Mészáros, Z.; Karádi, I.; Csányi, A.; Szombathy, T.; Romics, L.; Magyar, K. Eur. J.
Drug Metab. Pharmacokinet. 1999, 24, 299.
Plasma protein binding ratio ð%Þ ¼ ð1 ꢂ fpÞ ꢁ 100
8. Boomsma, F.; van Veldhuisen, D. J.; de Kam, P. J.; Man in’t Veld, A. J.; Mosterd,
A.; Lie, K. I.; Schalekamp, M. A. D. H. Cardiovasc. Res. 1997, 33, 387.
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fp ¼ Cf =Cp
where ‘Cf’ is the concentration of test compound in a PBS sample
after dialysis, and ‘Cp’ is the concentration of test compound in a
plasma sample after dialysis.
11. (a) Smith, D.; Vainio, P.; Mikkola, J.; Vuorio, P.; Vainio, J. WO 2008129124,
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(last
Acknowledgements
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Tavares, L. D.; Mattes, J.; Teixeira, M. M.; Jarolimek, W. Respir. Res. 2015, 16, 42;
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The authors thank the staff of the Astellas Research Technolo-
gies Co., Ltd. for conducting PAMPA assays, elemental analysis,
and spectral measurements. The authors also thank Mr. Kyoichi
Maeno and Dr. Susumu Watanuki for their valuable advice in the
preparation of this manuscript and Dr. Toshio Okazaki for his
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